Kinetic assays are performed by making measurements at a series of points in time to measure the change of a sample. The measurements at any one time point might also be used for a non-kinetic assay, here called a fixed endpoint assay. Fixed endpoint assays are sufficient for samples that exhibit little or no change over the duration of the assay. If the sample changes over time, kinetic measurements are required to measure those changes. Mathematical descriptions of the trends in various cell parameters over time represent kinetic features that are distinct from the measurements calculated in fixed endpoint assays.
Kinetic assays are performed on the same sample over time and are distinct from common experiments that provide an approximation of kinetic features with fixed endpoint assays on different portions of a sample. For example, if the sample is a population of cells comprising a number of similar individual cells, changes in the population over time can be measured by assaying portions of the sample with a series of fixed endpoint assays. This approach is commonly used in biochemical or immunohistochemical assays when samples are killed (i.e., fixed) or destroyed during the assay. A series of fixed endpoint assays makes measurements on individual cells, but the particular individuals within each population are different at each fixed endpoint assay and cannot be related to each other on the cell level. A series of fixed endpoint assays provides useful kinetic information only when the population average measurements are assumed to be related from portion to portion of the sample and the individual cells in the population are assumed to be equivalent.
The fixed endpoint approach is insufficient if the cells in the sample are not equivalent or if the changes must be related over time on a cell-by-cell basis. Measurements of physiologically relevant cells are heterogeneous, reflecting the normal variability of cell behavior in an intact animal. The heterogeneity often includes important information on the physiology of cells in the living state, and biologically relevant measurements must include, not exclude the variability of the sample. Living cells that change independently of each other must be measured at multiple times and the measurements correlated over time on a cell-by-cell basis.
A true kinetic assay addresses problems by providing measurements on single cells correlated through time. Generally, cells are identified by position and by other characteristics to provide continuity of cell level biological measurement at each time. A typical problem to be overcome is positional uncertainty of cells due to movement of cells or the measuring instrument. The ability to identify cells over time allows the user to measure and account for sample variability, and subpopulation behavior. The whole population response of a sample is often due to the activity of just a subpopulation of cells. Accurate kinetic measurement of subpopulations provides higher content information about physiological, or pharmacological response of a biological sample. Cell-based kinetic measurements also allow multiple measurements of the same sample (multiparametric assays) to be correlated on the cell level, connecting measurements of different cellular functions and mechanisms, and thus providing a better mechanistic understanding of cells and drugs that affect them.
Therefore, methods for tracking individual cells during a kinetic cell screening assay are needed in the art.